Variant lipolytic ensymes

ABSTRACT

The inventors have developed improved polypeptides by substituting or deleting specified amino acids in fungal lipolytic enzymes. More particularly, the polypeptides result in a reduction of dough stickiness when they are added to a dough. The polypeptides may particularly have activity on polar lipids.

FIELD OF INVENTION

The present invention relates to variant polypeptides made by alteringthe amino acid sequence of a fungal lipolytic enzyme, particularly tosuch polypeptides with improved properties for use in a dough, e.g. formaking bread and other baked products, and more particularly to suchpolypeptides having hydrolytic activity towards ester bonds in polarlipids.

BACKGROUND OF THE INVENTION

Phospholipases and galactolipases are known as enzymes with hydrolyticactivity towards ester bonds in polar lipids such as phospholipids andgalactolipids. WO 0032758 discloses lipolytic enzyme variants havingphospholipase and galactolipase activity and their use in baking. WO9826057 discloses a lipase/phospholipase from Fusarium oxysporum and itsuse in baking. WO 0183770 describes variants of a fungal lipase.

SUMMARY OF THE INVENTION

The inventors have developed variant polypeptides by modifying the aminoacid sequence of a parent polypeptide which is a fungal lipolyticenzymes. The variant polypeptides result in a reduced dough stickiness,compared to the parent polypeptide, when they are added to a dough.

Accordingly, the invention provides a method of producing a polypeptide,comprising:

a) selecting an amino acid sequence for a parent polypeptide which is afungal lipolytic enzyme,

b) selecting an amino acid residue in the sequence which corresponds toA29, K33, I83 or A255 of SEQ ID NO: 1 (corresponding to P29, N33, R84 orP256 of SEQ ID NO: 2),

c) modifying the amino acid sequence by substituting or deleting theselected residue,

d) preparing a variant polypeptide having the modified amino acidsequence, and

e) adding the polypeptide to a dough and testing dough stickiness.

The invention also provides a variant polypeptide which:

a) has hydrolytic activity towards an ester bonds in a polar lipid, and

b) has an amino acid sequence which

-   -   i) has at least 80% identity to SEQ ID NO: 1 and has a different        amino acid or an amino acid deletion at a position corresponding        to A29, K33, I83 or A255, or    -   ii) has at least 80% identity to SEQ ID NO: 2 and has a        different amino acid or an amino acid deletion at a position        corresponding to R84 or P256.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of amino acid sequences of fungal lipolyticenzymes to identify corresponding amino acids in SEQ ID NO: 1 to 15. SEQID NO: 1 is the lipase/phospholipase from Fusarium oxysporum (WO9826057). SEQ ID NO: 2 is a variant with pohospholipase andgalactolipase activity disclosed in WO 0032758. SEQ ID NO: 3 to 15 areknown lipolytic enzymes from the following organisms: Absidia reflexa,Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar (oryzae),Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum,Aspergillus oryzae, Penicilium camemberti, Aspergillus foetidus,Aspergillus niger, Aspergillus oryzae and Thermomyces lanuginosus.

DETAILED DESCRIPTION OF THE INVENTION

Parent Polypeptide

The parent polypeptide may have the sequence SEQ ID NO: 1 or 2 or onewhich can be aligned with SEQ ID NO: 1 or 2. It may have at least 50%amino acid identity to SEQ ID NO: 1 or 2, e.g. at least 60%, at least70% or at least 80%. Examples are the polypeptides having the sequencesSEQ ID NO: 1 to 14 or a variant disclosed in WO 0032758.

The parent polypeptide has lipolytic enzyme activity, e.g. hydrolyticactivity towards an ester bond in a polar lipid.

Variant Polypeptide

The amino acid at the position corresponding to A29 in SEQ ID NO: 1 maybe P. The amino acid at the position corresponding to K33 in SEQ ID NO:1 may be N. The amino acid at the position corresponding to 183 of SEQID NO: 1 may be A/R/N/D/C/Q/E/G/H/L/K/M/F/P/S/T/Y/V. The amino acid atthe position corresponding to A255 in SEQ ID NO: 1 may beR/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V.

The amino acid at the position corresponding to R84 of SEQ ID NO: 2 maybe A/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/Y/V. The amino acid at the positioncorresponding to P256 in SEQ ID NO: 2 may beA/R/N/D/C/Q/E/G/H/I/L/K/M/F/S/T/W/Y/V. The polypeptide may comprisefurther modifications compared to SEQ ID NO: 2., e.g. as disclosed in WO0032758. Thus, it may have the amino acid A/T at position D62, G/T atposition A91, D/F/S/G at position W96, E at position K99, G at positionS158, D at position G240, S at position N247, D at position N248, K/R atposition Q249, K/T at position P250, T at position N251, F at positionI252, M/R at position P253, S/Y/W at position D254, L at position I255,G at position A257, H/C at position W260, G at position Q263, L atposition A264, I at position T265, G/S/A at position D266, T at positionA267, L at position N269 and/or truncation after N269.

The polypeptide may additionally comprise amino acid modifications suchas insertions or deletions. Also, the N- or C-terminus may be modified,e.g. by truncating residues in SEQ ID NO: 2 after position 269 or byextending the C-terminal of SEQ ID NO: 2 withWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS. The C-terminal may betruncated after position 272, 273, 274 or 286 in SEQ ID NO: 1. TheN-terminal may have a peptide extension, e.g. as described in WO 0032758or WO 9704079, such as the addition of the amino acid residues SPIRR.

A similar amino acid substitution or deletion may be made in otherfungal lipolytic enzymes, e.g. SEQ ID NO: 3-14 at a correspondingposition. The corresponding positions may be found by aligning a givensequence with SEQ ID NO: 1 or 2, e.g. as shown in FIG. 1. The alignmentmay be done by use of the GAP program as described below.

The variant polypeptide may have improved thermostability compared tothe parent polypeptide, particularly a variant polypeptide having asubstitution at a position corresponding to A29 or K33 of SEQ ID NO: 1,e.g. the substitution A29P or K33N.

Sequence Identity

The variant polypeptide has at least 80% identity to SEQ ID NO: 1 or 2,particularly at least 85%, at least 90%, at least 95%, or at least 98%.The degree of identity between two sequences may be suitably determinedby means of computer programs known in the art, such as GAP provided inthe GCG program package (Program Manual for the Wisconsin Package,Version 8, August 1994, Genetics Computer Group, 575 Science Drive,Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970),Journal of Molecular Biology, 48, 443-45), using GAP with the followingsettings for polypeptide sequence comparison: GAP creation penalty of3.0 and GAP extension penalty of 0.1.

Dough Stickiness

The variant polypeptide may be tested by adding it to a dough andevaluating the dough stickiness. The dough may be generated according toa typical European straight dough procedure, a typical American sponge &dough procedure or any other bread making procedures. The polypeptidemay be added at a dosage of 0.01-10 mg enzyme protein per kg flour, andthe dough stickiness may be evaluated directly after mixing or at anypoint during processing. Of particular importance is the doughstickiness of the finally mixed dough, i.e. at the time where the doughruns through processing equipment such as divider, molder, sheeter andconveyer belts. The mixing time varies depending on procedure. For atypical European straight dough procedure, the mixing time can e.g. bein the range of 6-10 minutes. For a typical American Sponge & doughprocedure the mixing time can e.g. be in the range of 6-20 minutes (onfinal dough). The dough may have a resting period of 5-20 min beforefurther processing, e.g. at 20-35° C. The dough stickiness may beevaluated by hand by trained bakers, by a sensory panel or byinstrumental measurements e.g. by the Chen-Hoseney dough stickiness rigdeveloped for Stable Micro Systems TA-XT2 texture analyser, commerciallyavailable from Brookfield Engineering Laboratories, Inc.

Hydrolytic Activity towards Ester Bonds in Polar Lipids

The parent and variant polypeptides have lipolytic enzyme activity, i.e.they have hydrolytic activity towards an ester bond and are classifiedin EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature(available at http://www.chem.qmw.ac.uk/iubmb/enzyme). Morespecifically, they have hydrolytic activity towards ester bonds in polarlipids so as to split off acyl groups at the sn-1 and/or sn-2 positionof polar lipids such as phospholipids and galactolipids. Accordingly,they may have phospholipase activity or galactolipase activity (EC3.1.1.26), e.g. phospholipase A1 activity (EC 3.1.1.32).

Phospholipase activity may be determined by known methods, e.g. the“monolayer phospholipase assay” or the plate assay described in WO0032758. Galactolipase activity may be determined with digalactosyldiglyceride as substrate, e.g. as described in WO 0032758.

Use of Polypeptide

The polypeptide may be added to a dough, and the dough may be used toprepare a steamed bread, a baked product (particularly bread), pasta ornoodles. The addition of the polypeptide may lead to improved doughstabilization, i.e. a larger loaf volume of the baked product and/or abetter shape retention and volume during processing and baking,particularly in a stressed system, e.g. in the case of over-proofing orover-mixing. It may also lead to a lower initial firmness and/or a moreuniform and fine crumb, improved crumb structure (finer crumb, thinnercell walls, more rounded cells), of the baked product, and it mayfurther improve dough properties, e.g. a less soft dough, higherelasticity and/or lower extensibility.

The process may be conducted in analogy with U.S. Pat. No. 5,578,489 orU.S. Pat. No. 6,077,336. In the case of un-proofed frozen dough thepolypeptides of the invention perform better than known lipolytic enzymevariants in terms of volume and crumb structure.

The polypeptide can be used in a process for making bread, comprisingadding the polypeptide to the ingredients of a dough, kneading the doughand baking the dough to make the bread. This can be done in analogy withU.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.), EP 426211 (Unilever) orWO 99/53769 (Novozymes).

The composition of a typical dough can be found in WO 99/53769.

The polypeptide of the invention may be added together with ananti-staling amylase and optionally also a phospholipid as described inWO 9953769, particularly a maltogenic alpha-amylase (e.g. from Bacillussp., such as Novamyl® from Novo Nordisk). Also, a fungal or bacterialα-amylase may be added, e.g. from Aspergillus or Bacillus, particularlyA. oryzae, B. licheniformis or B. amyloliquefaciens. Optionally anadditional enzyme may be added, e.g. an amyloglucosidase, abeta-amylase, a pentosanase such as a xylanase as described in WO99/53769, e.g. derived from Aspergillus, in particular of A. aculeatus,A. niger (cf. WO 91/19782), A. awamori (WO 91/18977), or A. tubigensis(WO 92/01793), from a strain of Trichoderma, e.g. T. reesei, or from astrain of Humicula, e.g. H. insolens (WO 92/17573), a proteiase and/or aglucose oxidase.

The dough may further comprise an emulsifier such as mono- ordiglycerides, diacyl tartaric acid esters of mono- or diglycerides,sugar esters of fatty acids, polyglycerol esters of fatty acids, lacticesters of monoglycerides, acetic acid esters of monoglycerides,polyoxyethylene stearates, polysorbates or lysolecithin.

The dough may also comprise other conventional dough ingredients, e.g.:proteins, such as milk powder, gluten, and soy; eggs (either whole eggs,egg yolks or egg whites); an oxidant such as ascorbic acid, potassiumbromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar; a salt such assodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

EXAMPLES

Baking Evaluation of Polypeptides with Phospholipase Activity

In the examples, polypeptides according to the invention were testedtogether with the corresponding parent polypeptide in a bakingevaluation experiment by using conventional baking protocols forEuropean straight dough procedure and US sponge & dough procedure, asfollows:

European Straight Dough Procedure:

A dough is prepared by mixing the below ingredients for 3 minutes slowand 7 minutes fast. % (baker's - by weight) Flour 100 Compressed yeast 4Salt 1.5 Sugar 1.5 Water 62 Ascorbic acid 40 ppm

Dough stickiness is evaluated right after mixing and again after aresting period of 15 minutes. Dough stickiness is evaluated by a trainedand experienced bakers by sensory evaluation by hand. Dough stickinessis a measure of how sticky the dough feels and is expressed on a scalefrom 0 (little stickiness) to 10 (very sticky). The dough with thevariant is compared to a reference dough, which is always given thescore 5.

Sponge & Dough Procedure: Sponge Dough % (baker's - by weight) %(baker's - by weight) Flour 60 40 Compressed yeast 7.5 Oil 2.5 Salt 2High fructose syrup 12 Water 34.4 20.4 Ascorbicc acid 50

A liquid sponge is prepared by mixing a sponge consisting of the abovelisted sponge ingredients for 1 minute slow and 4 minutes fast. Thesponge is fermented for 3 hours at 27 C, 86% RH. The sponge is mixedwith the dough ingredients listed above and with enzymes for 1 minutesslow and 18 minutes fast.

Dough stickiness is evaluated right after mixing, whereafter the doughis extruded on a rebuild pasta-machine to simulate the dough extrusionused for dough dividing in US. Dough stickiness is evaluated again afterextrusion. Dough stickiness is evaluated by a trained and experiencedbakers by sensory evaluation by hand. Dough stickiness is a measure ofhow sticky the dough feels and is expressed on a scale from 0 (littlestickiness) to 10 (very sticky). The dough with the variant polypeptideis compared to a reference dough made with the parent polypeptide, whichis always given the score 5.

Example 1 Construction of Polypeptides

Polypeptides according to the invention were prepared as described in WO00/32758. The polypeptides were derived from SEQ ID NO: 15 by making thefollowing amino acid modifications. Polypeptide Amino acid alterationscompared to SEQ ID NO: 15 1 G91A +D96W +E99K +P256M +G263Q +L264A +I265T+G266D +T267A +L269N +270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 2 G91A +D96W +E99K+P256N +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G +273F+274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 3 G91A +D96W +E99K+P256V +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G +273F+274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 4 G91A +D96W +E99K+N247S +N248D +Q249K +N251T +P253M +D254S +P256L +A257A +G263Q +L264A+I265T +G266D +T267A +L269N 5 G91A +D96W +E99K +N247S +N248D +Q249R+P250T +N251T +P253M +D254W +P256V +A257G +G263Q +L264A +I265T +G266D+T267A +L269N 6 G91A +D96W +E99K +P256T +G263Q +L264A +I265T +G266D+T267A +L269N +270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 7 G91A +D96W +E99K+P256A +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G +273F+274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 8 G91A +D96W +E99K+G240D +P256C +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G+272G +273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 9 G91A+D96W +E99K +P256G +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G+272G +273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 10G91A +D96W +E99K +P256R +G263Q +L264A +I265T +G266D +T267A +L269N +270A+271G +272G +273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS11 G91A +D96W +E99K +P256Q +G263Q +L264A +I265T +G266D +T267A +L269N+270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 12 G91A +D96W +E99K+P256K +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G +273F+274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 13 G91A +D96W+E99K +P256L +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G+273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 14 G91A+D96W +E99K +P256D +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G+272G +273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 15R84E +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +T267A +L269N+270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 16 R84M +G91A +D96W+E99K +P256V +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +272G+273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 17 R84P+G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +T267A +L269N +270A+271G +272G +273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS18 R84S +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +T267A+L269N +270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 19 G91A +D96W +E99K+P250K +N251T +I252F +P253R +D254Y +I255L +P256del +G263Q +L264A +I265T+G266D +T267A +L269N +270A +271G +272G +273F +274S+275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

Example 2 Baking Evaluation of a Polypeptide According to the Invention

5 variant polypeptides according to the invention were compared to theparent polypeptide (SEQ ID NO: 2) in the European straight doughprocedure described above. 40 ppm Fungamyl Super MA (a blend of fungalalpha-amylase and xylanase) was added as background to all doughs. Theparent enzyme and the variants were dosed at their optimal level, i.e.the level giving best volume and dough stabilising effect. The belowresults show that all 5 variants give reduced dough stickiness comparedto the parent polypeptide. Polypeptide Parent P256V P256A P256Q P256LP256W Dough 6 5 5 5 5 5 stickiness after mixing Dough 6 5 5 5 5 5stickiness after 15 min table time

3 variant polypeptides according to the invention were compared to theparent polypeptide (SEQ ID NO: 1) in the European straight doughprocedure described above. 40 ppm Fungamyl Super MA (a blend of fungalalpha-amylase and xylanase) was added as background to all doughs. Theparent enzyme and the variants were dosed at their optimal level, i.e.the level giving best volume and dough stabilising effect. The belowresults show that all 4 variants give reduced dough stickiness comparedto the parent enzyme Polypeptide Parent A29P+K33N+I83T A29P+K33N+I83HA29P+K33N+I83Q Dough stickiness 5 4 4 4 after mixing Dough stickiness 54 4 4 after 15 min table time

4 variant polypeptides according to the invention were compared to theparent enzyme (SEQ ID NO: 1) in the European straight dough proceduredescribed above. 10FAU Fungamyl/kg was added as background to alldoughs. The parent enzyme and the variants were dosed at their optimallevel, i.e. the level giving best volume and dough stabilising effect.The below results show that all 4 variants give reduced dough stickinesscompared to the parent enzyme Polypeptide Parent A29P + K33N A29P + I83NK33N + I83E K33N + I83K Dough stickiness 7 6 6 6 6 after mixing Doughstickiness 7 6 6 6 6 after 15 min table time

A variant polypeptide according to the invention was compared to itsparent enzyme (SEQ ID NO: 1) in the US sponge & dough proceduredescribed above. 40 ppm Fungamyl Super MA (a blend of fungalalpha-amylase and xylanase) was added as background to all doughs. Theparent enzyme and the variant were dosed at their optimal level, i.e.the level giving best volume and dough stabilising effect. The belowresults show that the variant gives reduced dough stickiness compared tothe parent enzyme Polypeptide Parent A29P+I83N Dough stickiness after 65 mixing Dough stickiness after 6.5 5 extrusion

Example 3 Variant Polypeptides Derived from SEQ ID NO: 1

Variant polypeptides with the following amino acid alterations comparedSEQ ID NO: 1 (lipase/phosapholipase from F. oxysporum) were prepared andtested by adding each polypeptide to a dough. The polypeptide withunmodified SEQ ID NO: 1 was also tested, for comparison. A29P K33N A29P+I83T A29P +I83N A29P +I83C A29P +I83F A29P +I83L K33N +I83W K33N +I83LK33N +I83Q K33N +I83S K33N +I83N K33N +I83N K33N +I83R K33N +I83L K33N+270VASLGDDTEAPRASTRGPP A29P +I83N +A255V

The results were that with each of the above polypeptides, doughstickiness was better than with the polypeptide with the unmodifiedsequence of SEQ ID NO: 1.

Baking tests with each dough showed that all polypeptides improved thecrumb structure, the loaf volume and the dough stability, both for themodified and unmodified sequences.

Example 4 Variant Polypeptides Derived from SEQ ID NO: 2

Variant polypeptides with the following amino acid alterations comparedSEQ ID NO: 2 (variant of T. lanuginosus lipase) were prepared and testedby adding each polypeptide to a dough. The polypeptide with unmodifiedSEQ ID NO: 2 was also tested for comparison. R84D R84I R84M R84Q P256AP256D P256I P256L P256Q P256S P256V

The results were that with each of the above polypeptides, doughstickiness was better than with the polypeptide with the unmodifiedsequence of SEQ ID NO: 2.

Baking tests with each dough showed that all polypeptides improved thecrumb structure, the loaf volume and the dough stability, both for themodified and unmodified sequences.

1-14. (canceled)
 15. A method of producing a polypeptide, comprising: a)selecting an amino acid sequence for a fungal lipolytic enzyme, b)selecting an amino acid residue in the sequence which corresponds toA29, K33, I83 or A255 of SEQ ID NO: 1, c) modifying the amino acidsequence by substituting or deleting the selected residue, d) preparinga polypeptide having the modified amino acid sequence, and e) adding thepolypeptide to a dough and testing dough stickiness.
 16. The method ofclaim 15, which further comprises testing hydrolytic activity of thepolypeptide towards ester bonds in polar lipids and selecting apolypeptide which has such activity.
 17. A method of producing a dough,comprising: a) preparing a variant lipolytic enzyme which comprises anamino acid sequence which has at least 80% identity to SEQ ID NO: 1 orSEQ ID NO:2 and a substitution or deletion of an amino acid residuewhich corresponds to amino acid residues A29, K33, I83 or A255 of SEQ IDNO: 1, and b) adding the variant lipolytic enzyme to a dough.
 18. Themethod of claim 17, wherein said method further comprises baking thedough.
 19. The method of claim 17, wherein said method comprisespreparing a variant lipolytic enzyme which comprises an amino acidsequence which has at least 85% identity to SEQ ID NO:1 or SEQ ID NO:2.20. The method of claim 17, wherein said method comprises preparing avariant lipolytic enzyme which comprises an amino acid sequence whichhas at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2.
 21. The methodof claim 17, wherein said method comprises preparing a variant lipolyticenzyme which comprises an amino acid sequence which has at least 95%identity to SEQ ID NO:1 or SEQ ID NO:2.
 22. The method of claim 17,wherein said method comprises preparing a variant lipolytic enzyme whichcomprises an amino acid sequence which has at least 98% identity to SEQID NO:1 or SEQ ID NO:2.
 23. The method of claim 17, wherein said variantlipolytic enzyme has phospholipase activity or galactolipase activity(EC 3.1.1.26).
 24. A polypeptide which has hydrolytic activity towardsan ester bond in a polar lipid, and comprises an amino acid sequencewhich i) has at least 80% identity to SEQ ID NO: 1 and has a differentamino acid as compared to a position corresponding to A29, K33, I83 orA255 of SEQ ID NO:1 or an amino acid deletion at a positioncorresponding to A29, K33, I83 or A255 of SEQ ID NO:1, or ii) has atleast 80% identity to SEQ ID NO: 2 and has a different amino acid ascompared to a position corresponding to A29, K33, I83 or A255 of SEQ IDNO:1 or an amino acid deletion at a position corresponding to R84 orP256 (using SEQ ID NO:1 for numbering).
 25. The polypeptide of claims24, wherein the polypeptide has phospholipase activity or galactolipaseactivity (EC 3.1.1.26).
 26. The polypeptide of claim 24, wherein theamino acid at the position corresponding to A29 of SEQ ID NO: 1 is P.27. The polypeptide of claim 24, wherein the amino acid at the positioncorresponding to K33 of SEQ ID NO: 1 is N.
 28. The polypeptide of claim24, wherein the amino acid at the position corresponding to I83 of SEQID NO: 1 is N/C/W.
 29. The polypeptide of claim 24, which consists ofamino acids 1-272, 1-273, 1-274 or 1-286 of SEQ ID NO: 1 with thefollowing substitutions: A29P K33N A29P +I83T A29P +I83N A29P +I83C A29P+I83F A29P +I83L K33N +I83W K33N +I83L K33N +I83Q K33N +I83S K33N +I83NK33N +I83R K33N +I83L K33N +270VASLGDDTEAPRASTRGPP A29P +I83N +A255V


30. The polypeptide of claim 24, wherein the amino acid at the positioncorresponding to R84 of SEQ ID NO: 2 is L/M/Q/I/D.
 31. The polypeptideof claim 24, wherein the amino acid at the position corresponding toP256 of SEQ ID NO: 2 is V/Q/A/D/S/I.
 32. The polypeptide of claim 24,wherein the polypeptide consists of SEQ ID NO: 2 with the followingsubstitutions: R84D R84I R84M R84Q P256A P256D P256I P256L P256Q P256SP256V


33. The polypeptide of any of claim 24, wherein said polypeptide ascompared to SEQ ID NO: 2 has the amino acid A/T at position D62, G/T atposition A91, D/F/S/G at position W96, E at position K99, G at positionS158, D at position G240, S at position N247, D at position N248, K/R atposition Q249, K/T at position P250, T at position N251, F at positionI252, M/R at position P253, S/Y/W at position D254, L at position I255,G at position A257, H/C at position W260, G at position Q263, L atposition A264, I at position T265, G/S/A at position D266, T at positionA267, L at position N269 and/or is truncated after N269.
 34. Apolynucleotide encoding the polypeptide of claim 26.